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Aggregation of microtubule initiation sites preceding neurite outgrowth in mouse neuroblastoma cells
Cell, Vol. 16,253-263,
February
1979,
Copyright
0 1979 by MIT
Aggregation of Microtubule Initiation Sites Preceding Neurite Outgrowth in Mouse Neuroblaktoma Cells Bruce M. Spiegelman,* Margaret A. Lopatat Marc W. KirschnertS Department of Biochemical Sciences Princeton University Princeton, New Jersey 08540
and
Summary By examining microtubule regrowth using immunofluorescence with antibody to tubulin, we have studied the structure and intracellular localization of microtubule initiation sites in undifferentiated and differentiated mouse neuroblastoma cells. The undifferentiated cells are round and lack cell processes. They contain an average of 12 initiation sites per cell. Each of these sites, which are located near the cell nucleus, initiates the growth of several microtubules in a radial formation. In contrast to the undifferentiated cells, neuroblastoma cells stimulated to differentiate by serum deprivation are asymmetrical, containing one or two very long neurites. These cells have a single, large microtubule initiation center which can be visualized not only by immunofluorescence but by phase-contrast and differential interference microscopy as well. The initiation site measures 3-4 p in diameter and is located in the cell body along a line defined by the neurite. During cell differentiation, the large initiation center seems to be formed by the aggregation of many smaller sites. This process precedes neurite extension by about 24 hr. The growth of microtubules from this center appears to be highly oriented, since most microtubules initially grow into the neurite processes rather than into the cell interior. Thus major changes in the structure and location of microtubule initiation sites occur during the differentiation of neuroblastoma cells. Similar changes are likely to be involved in alterations in the morphology of other cell types. Introduction Changes in cell shape are important in embryonic development and cell differentiation. The arrangement of structural proteins, such as actin, microtubules and intermediate filaments, strongly influences if not determines cell morphology. We may therefore expect that changes in the intracellular distribution of these structural elements are a sigl Present address: Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139. t Present address: Department of Biochemistry and Biophysics, University of California School of Medicine, San Francisco, California 94143. $ To whom correspondence should be addressed.
nificant part of cell differentiation. For example, Perry and Waddington (1966) showed that microtubules are prominent components of the necks of the highly asymmetric bottle cells present at the blastopore in amphibians. They are also present in elongating cells in the primitive streak in birds (Granholm and Baker, 1970). Microtubules are particularly prominent in neural development. The highly asymmetric nerve processes are packed with dense parallel arrays of microtubules. Treatment of immature nerve cells with antimicrotubule drugs such as colchicine prevents neurite extension and causes withdrawal of previously extended neurites (Yamada, Spooner and Wessels, 1970; Seeds et al., 1970; Daniels, 1975), indicating that microtubules are required for both extension and maintenance of these cell processes. The mechanism by which the cell changes its microtubule distribution is unknown. However, since microtubules grow from distinct initiation centers (Frankel, 1976; Osborn and Weber, 1976; Brinkley, Fuller and Highfield, 1976; Spiegelman, Lopata and Kirschner, 1979), the precise location and structure of these centers may determine the microtubule distribution and hence the cell morphology. In the accompanying paper (Spiegelman et al., 1979), we have shown that mammalian fibroblasts and epithelial cells contain several initiation sites for microtubule growth. Three classes of sites could be distinguished and their arrangement and nature were found to differ in epithelial and fibroblast cells. In this study, we have sought to determine whether the position of these sites is closely related to cell morphology and whether changes in initiation sites accompany cellular differentiation. We have compared the structure and distribution of these sites in neuroblastoma cells before, during and after stimulation of neurite outgrowth. The results show that initiation sites alter their position and coalesce before the process of neurite outgrowth, suggesting that changes in their arrangement may control neurite formation. Results lmmunofluorescence of Neuroblastoma Cells with Antibody to Tubulin When grown in culture medium containing 10% serum, mouse neuroblastoma cells (line N11.5) are small rounded cells, 30-50 p in diameter, having few extensions or processes (Figure IA). Removal of serum causes cell differentiation (Seeds et al., 1970). Figure 1C shows the effect of 5 days of incubation in culture medium lacking serum. Approximately 80-90% of the cells have extended neurites which may be as long as 500 p. Most of
Cell 254
the cells contain a single neurite, but we occasionally observed cells with two neurites, such as one of those shown in Figure 1C. Accompanying this morphological change induced by serum deprivation are a number of enzymatic and electrical changes (Augusti-Tocco and Sato, 1969; Nelson, Ruffner and Nierenberg, 1969; Blume et al., 1970), which suggest that this system may be a model for nerve cell differentiation. To visualize microtubule networks in the neuroblastoma cells, we have stained cells with antibody to tubulin with the same cytoskeleton preparation technique used in our previous study of fibroblast and epithelial cells (Spiegelman et al., 1979, adapted from Osborn and Weber, 1977). lmmunofluorescence staining of undifferentiated cells and of cells induced to differentiate by serum deprivation is shown in Figures 1 B and lD, respectively. In both the undifferentiated and differentiated cells, intense staining is observed throughout the cells. It appears that the interphase microtubule network is so thick that it is difficult to resolve individual microtubules. Only in exceptionally flat cells can distinct microtubules be seen at the cell periphery (see also Osborn and Weber, 1977; Isenberg, Rieske and Kreutzberg, 1977). Microtubule Regrowth in Undifferentiated Neuroblastoma Cells As we observed in fibroblast and epithelial cells (Spiegelman et al., 1979), microtubule initiation sites in neuroblastoma cells are readily visualized during the process of microtubule regrowth after exposure to depolymerizing agents. If cells are exposed to colcemid for 90 min at 0.4 pg/ml, no microtubule structures are visible. When cells are exposed to colcemid for 35 min and then allowed to recover for 25 min in fresh medium, several bright points of fluorescence are visible in the interior of the cell, as shown in Figure 2A. Figure 2B shows the phase-contrast image of the same cell. We could not identify any distinct structures in phase-contrast which correspond to the bright fluorescent points. After IO min of further incubation in the absence of the drug, microtubules grow extensively from several sites. In Figure 2C, nine sites are visible surrounding the cell nucleus. Figure 3 is a higher magnification photograph of another undifferentiated cell, showing regrowth from 14 distinct sites in the perinuclear region. The microtubules appear to grow out in all directions from the individual initiation points. After 50 min of recovery, microtubules have grown throughout the cell, although the initiation sites are still discernible, as shown in Figure 2D. After this time, individual microtubules are difficult to resolve, presumably because there is a superposition of dense
arrays of microtubules. We find that undifferentiated neuroblastoma cells contain an average of 12.3 i- 4.5 initiation sites per cell, with a range of 6-27. in 43 cells. Microtubule Regrowth in Differentiated Neuroblastoma Cells There are special problems associated with observing microtubule regrowth in differentiated neuroblastoma cells. As Seeds et al. (1970) showed, cellular processes are extremely sensitive to antimicrotubule drugs. We have found, for example, that exposure of cells to colcemid at 0.4 pglml for 90 min is sufficient to cause complete absorption of neurites into the cell body and eliminates immunofluorescent staining of any discrete microtubule structures. Very short exposures, however, will achieve nearly complete microtubule depolymerization without neurite retraction. Since we have found that microtubule depolymerization continues for about 25 min after the removal of colcemid, we have minimized the treatment times by exposing cells for a period short enough so that depolymerization approaches completion only during the recovery period. Thus the length of time the cell is devoid of its microtubule network is kept to a minimum. When differentiated neuroblastoma cells are exposed to 0.4 pg/ml of colcemid for 35 min, followed by 25 min of recovery in fresh medium, the neurites are retained, but the bright fluorescence disappears from the neurite and most of the cell body. A single fluorescent center, however, is visible in these cells (Figure 4A). In virtually all cases, this center is found in the cell body along a line defined by the neurite extension. It occurs most commonly between the neurite and the cell nucleus but occasionally can be found directly on top of the cell nucleus. Although it is difficult to see in a photograph taken at a single focal plane, this structure has an irregular scalloped appearance and seems to be an aggregate of a number of fluorescent points. After 10 min of further incubation in colcemid-free medium, short microtubules can be seen emanating in many directions in a roughly radial fashion from the large fluorescent center (Figure 4C). Unlike fibroblasts, neuroblastomas are thick cells and the microtubules do not all lie in the same plane of focus, making them more difficult to photograph. Figure 4D shows that after 50 min in colcemid-free medium, the microtubules have elongated into the neurite. At this stage of regrowth, the microtubules seem oriented, since less growth extends into the cell body than into the neurite. After longer periods of recovery, the intensity of fluorescence increases throughout the cell and the neurite, and the detailed microtu-
Aggregation 255
Figure
of Microtubule
1. lmmunofluorescence
Initiation
Sites
of Mouse
Neuroblastoma
Cells with Antibody
to Tubulin
Cells were grown on glass coverslips and induced to differentiate by 5 days of serum immunofluorescence by the cytoskeletal procedure of Osborn and Weber (1977) as described (A) (275X) phase-contrast image of undifferentiated cells: (B) (770X) immunofluorescence contrast image of differentiated cells; (D) (770X) immunofluorescence of a differentiated cell.
starvation. They were then processed for by Spiegelman et al. (1979). Bar = IO p. of undifferentiated cells: (C) (275X) phase-
Cell 256
Figure
2. Regrowth
of Microtubules
in Undifferentiated
Neuroblastoma
Cells
after Colcemid
Treatment
Cells grown on coverslips were treated with 0.4 pg/ml colcemid for 35 min, rinsed 3 times with immunofluorescence at the indicated times after the removal of colcemid. Bar = IO CL. (A) (770X) 25 min; (6) (770X) phase-contrast of same cell as (A); (C) (770X) 35 min; (D) (770X) 50 min.
fresh
medium
and
processed
for
Aggregation 257
Figure
of Microtubule
3. Higher
Ceils were
treated
Magnification as described
Initiation
Sites
Photograph in the legend
of Microtubule to Figure
Regrowth
2. Recovery
bule array is obscured, as in Figure 1D. In over 1000 cells examined, those containing neurite processes always contained asingle large initiation center. An image corresponding to the large microtubule initiation center can be observed with phase-contrast and differential interference microscopy in cytoskeletal preparations of differentiated neuroblastoma cells. An initiation structure is shown in Figure 4A with immunofluorescence. The corresponding structure is shown in the phase-contrast photograph of the same cell in Figure 4B, where it appears as a single, irregularly shaped dark object 3-4 p in diameter. In Figure 5B, a differentiated neuroblastoma cell is shown by differential interference microscopy. The initiation center appears to be a roughly circular structure 3-4 p in diameter, adjacent to the cell nucleus, which corresponds in size and position to the initiation structure stained with the fluorescent antibody (Figure 5A). The differential interference image shows that the initiation center is nonuniform and appears to be composed of several discrete components. The size and shape of the initiation center seen by
in Undifferentiated after
the removal
Neuroblastoma of colcemid
Ceils
was for 35,min
(1760X).
Bar = IO p.
phase-contrast and differential interference microscopy remain unchanged throughout the initial stages of microtubule regrowth. About 10% of the differentiated neuroblastoma cells contain two neurite processes. These cells also contain only a single large microtubule initiation center. Figure 6 shows a cell with two neurites which was allowed to recover for 50 min after treatment with colcemid. The large fluorescent center is located above the nucleus. Microtubule regrowth is bidirectional in this case, with most of the microtubules growing into the two neurites. Very few microtubules grow perpendicular to a line connecting the two neurites and the initiation structure. We observed similar patterns of regrowth to those observed with colcemid after treatment with vinblastine in both differentiated and undifferentiated cells. Our attempts to examine microtubule regrowth in neuroblastoma cells after griseofulvin treatment have been unsuccessful. In differentiated cells, we could not achieve sufficient depolymerization without the neurites regressing into the cell body. In undifferentiated cells, the background
Cell 258
Figure 4. Regrowth of Microtubules Differentiated Neuroblastoma Cells
after
Colcemid
Cells were grown on coverslips in serum-free Theywere then treated with 0.4 kg/ml colcemid 3 times with fresh medium and processed cence at the indicated times after the removal IO/L. (A) (578X) 25 min; (B) (578X) phase-contrast (C) (578X) 35 min; (D) (578X) 50 min.
Treatment
in
medium for 3 days. for35 min, rinsed for immunofluoresof colcemid. Bar = of same
cell as (A);
staining after griseofulvin treatment was too tense to allow clear examination of regrowth.
in-
Time Dependence of Initiation Site Aggregation and Neurite Outgrowth It is not clear from these immunofluorescence experiments whether the formation of the single large initiation center in the differentiated neuroblastoma cells precedes or follows the extension of
Figure 5. Differential Interference rescence of an Initiation Center Cells
Microscopy in Differentiated
and ImmunofluoNeuroblastoma
Cells were treated as described in the legend to Figure 4 and photographed 35 min after the removal of colcemid. Bar = 10 CL. (A) (1160X) immunofluorescence; (6) (1160X) differential interference image of the same cell as (A).
neurites. To answer this question, we have followed the time course of neurite outgrowth and the formation of single large sites in the same cultures. Undifferentiated neuroblastoma cells were initially
Aggregation
of Microtubule
Initiation
Sites
259
Table 1. Time Course of Appearance Aggregation of Initiation Sites
of Neurites
and
(A)
(‘3
CC)
P)
W
Days after Removal of Serum
Cells with Neurites
Dispersed Sites
Aggregated Sites
Single Large Center
w
w
(“W
(“4
0
2
90
0.5
9.5
1
7
05
6
9.0
2
8
35
33
3
58
18
10
72
4
76
12
16
72
5
81
4
6
90
32
Cells on coverslips were grown in serum-free medium for the indicated length of time and examined for the presence of neurites of any length by phase-contrast microscopy. The same coverslips were then processed for immunofluorescence by the cytoskeletal procedure after treatment of cells with 0.4 wg/ml colcemid and 35 min of recovery incubation in fresh medium at 37°C. Cells which had a single large microtubule initiation structure were scored. Cells which contained aggregates of three or more distinct initiation sites, but which had not formed a single large initiation structure, were scored as “aggregated.” For each time point, 250-300 cells were examined.
Figure 6. Regrowth Two Neurites
of Microtubules
Cells were treated as described processed for immunofluorescence colcemid (770X). Bar = 10 /I.
in a Neuroblastoma
Cell with
in the legend to Figure 4 and 50 min after the removal of
grown on coverslips in medium containing 10% fetal calf serum, placed in serum-free medium and then examined at various times for neurite formation. The cells showing neurites of any size were scored. These cells were then treated with colcemid for 35 min, allowed to recover for 35 min and processed for immunofluorescence to visualize the initiation centers. We noted the number of cells with single initiation centers and examined whether these cells had neurites. For each time point, 250-300 cells were examined. As shown in Table 1, only 1.8% of the neuroblastoma cells grown in medium containing serum had neurites, while 9.5% had single initiation centers. At all time points, every cell with neurites had a single initiation center. After 1 day without serum, the number of cells with neurites grew to 7%, while the number of cells with single initiation centers remained about the same at 9%. At 2 days, there was a greater than 3 fold increase in the proportion
of cells with single initiation centers to 32%, while the percentage of cells with neurites increased only slightly to 8%. At 3 days, there was a large proliferation of cells with neurites, increasing 7 fold to 58%, and an increase in the number of cells with single centers to 72%. Thereafter the percentage of cells with neurites and cells with single centers increased more slowly. In all cases, the number of cells with neurites was a subset of the cells with single initiation centers. Although one must be cautious in interpreting kinetic data, the analysis of initiation sites and neurites is clearly inconsistent with the notion that neurite outgrowth precedes the formation of a single large center. At 2 days, 4 times as many cells had single large centers as had neurites and with time this difference narrowed. There were no clear examples of cells with neurites that did not have a single initiation center. A replotting of columns B and E of Table 1 (not shown) shows that the time course of appearance of neurites is displaced about 24 hr after the appearance of single initiation sites. Intermediates in Initiation Site Aggregation The kinetic data suggest that the appearance of single large initiation centers precedes neurite formation. They do not, however, suggest how this occurs. We have surveyed the neuroblastoma cells during serum deprivation and have noted that in
Cell 260
reaching 90% at 5 days. This behavior suggests that small aggregates of initiation sites are intermediates in the formation of single, large complex centers.
cultures deprived of serum for 2 days, many cells contain multiple initiation sites which appear to be in the process of aggregating. Three such cells, which lack neurites but show initiation in various stages of aggregation, are shown in Figure 7. The appearance of the initiation centers in these cells should be contrasted with the typical dispersed appearance of the initiation sites in undifferentiated cells in Figures 2 and 3. In Table 1, we have distinguished among cells containing dispersed sites (column C), cells containing aggregates of at least three initiation sites (column D) and cells containing a single large initiation center (column E). At time zero, 90% of the cells had dispersed sites; a very small percentage (0.5%) had aggregated sites, while 9.5% had single, large centers. By the second day of serum deprivation, about one third of the cells had dispersed sites, one third had aggregated sites and one third had single initiation centers. Thereafter the number of cells containing aggregated sites dropped to 6% at day 5, while the number of cells with single centers increased to 90%. Thus small aggregates of initiation sites occur in a large portion of cells at 2 days, but are less evident at 0 and 5 days. At the same time, the number of cells with single centers increases markedly at 2 and 3 days,
Figure
7. Intermediates
Cells grown min recovery
on coverslips incubation
in the Aggregation were incubated in fresh medium,
of Microtubule
Initiation
Discussion In the accompanying paper (Spiegelman et al., 1979) we showed that mammalian epithelial and fibroblast cells have several initiation sites for microtubule assembly within each cell. This paper examines the number and nature of these sites during a developmental process thought to involve microtubule assembly. Although many cellular differentiation events have been shown to involve microtubules, the study of neurite outgrowth in neuroblastoma cells has particular advantages. Neurite outgrowth depends completely upon the assembly of microtubules. In addition, neuroblastoma differentiation is readily initiated in culture, takes place in single cells rather than in whole tissues and seems to be related to the normal process of nerve growth (Seeds et al., 1970). Changes in Initiation Sites during Differentiation Undifferentiated neuroblastoma cells have several (12 +- 4) sites for microtubule initiation in each cell.
Sites
in serum-free medium for 2 days. After treatment with 0.4 pg/ml colcemid they were processed for immunofluorescence (770X). Bar = 10 CL.
for 35 min and a 35
Aggregation 261
of Microtubule
Initiation
Sites
These multiple sites are observed when visualized after depolymerization by colcemid or vinblastine. They are rather uniformly arranged around the cell nucleus and there is no apparent clustering of sites. Similarly, we observed no clustering of initiation sites in fibroblast or epithelial cells (Spiegelman et al., 1979). None of these cell types is highly polarized or asymmetric and all contain uniformly arranged initiation sites, suggesting that the arrangement of these sites may be related to the overall cell morphology. In contrast to the undifferentiated cells, the differentiated neuroblastoma cells are highly asymmetric, possessing one or sometimes two long neurites. Differentiated cells have only one initiation center per cell, which is located near the cell nucleus, directly on a line from the neurite process. We observed a single initiation center in all cells which had neurite processes. However, not all cells which had single initiation centers had neurites. As shown in Table 1, 2% of the undifferentiated cells had neurites but 7% had single initiation centers. The disparity is even more striking 2 days after the removal of serum, when 8% of the cells had neurites and 32% had single initiation centers. Thus it seems very improbable that single large initiation centers could be caused by neurite formation, and more plausible that neurite production was a consequence of the formation of single initiation sites. This problem was studied more extensively by examining the time course of neurite outgrowth and the aggregation of initiation sites. The time course experiments in Table 1 demonstrate that the formation of a single large center precedes neurite formation and suggests that the formation of this large center occurs by aggregation of the dispersed sites. The fraction of cells with neurites and the fraction of cells with single centers both increase monotonically with time, while the fraction of cells with dispersed sites decreases monotonically. A plot of the data indicates that neurite extension lags about 24 hr behind the formation of single initiation centers. The fraction of cells with aggregated initiation sites shows the behavior of a kinetic intermediate, increasing from 0.8 to 33% from 0 to 2 days, and thereafter decreasing to 6% 5 days after the removal of serum. The peak in the fraction of cells with aggregated sites occurs before the rapid increase in cells with neurites. It was possible to see some structure in the large initiation center using immunofluorescence, although the structure was difficult to illustrate by photographs taken at a single plane of focus. The initiation center appears to be an irregular mass which might represent an aggregate of individual sites. Phase-contrast microscopy of cytoskeletal
preparations shows a dense body having the same position, size and shape as that seen by immunofluorescence (see Figures 4A and 4B). The differential interference image of this center in Figure 58 also gives the impression of a compound structure about 3 p in diameter. There is probably little more that can be learned about the structure of the large initiation center by light microscopy: further information will certainly require electron microscopic investigations. There are several reasons to suspect that neurite extension requires one or more steps in addition to the aggregation of microtubule initiation sites. First, there is a significant time lag of about 24 hr between the two processes, which allows the observation of many cells lacking neurites but containing aggregated initiation sites. Such cells would be expected to be rare if the aggregation of initiation sites was the last step or the rate-limiting step in neurite formation. Second, the first microtubules to regrow from the initiation center in differentiated cells seem to be directed into the neurite. This result is in contrast to the undirected growth from the dispersed sites in undifferentiated cells (Figures 4 and 6), and suggests that there must be a mechanism for orienting microtubules as well as a mechanism for localizing their points of initiation. This could be accomplished in several ways, such as assembly of the initiation center in a manner which allows microtubule growth in only one direction. Alternatively, there may be localized structures on the cell membrane which would stabilize certain microtubules or promote their assembly. A model for neurite outgrowth in neuroblastoma cells summarizing these observations is shown in Figure 8. The undifferentiated cell contains several initiation sites dispersed around the cell nucleus. Steps 1 and 2 illustrate the migration of initiation sites into one part of the cell and their aggregation into a single complex center. In step 3, microtubules are oriented, through some unknown mechanism, so that they grow primarily in a single direction toward the cell membrane. In steps 4 and 5, neurites grow out from the cell body and elongate. This process depends upon the polymerization of microtubules. If neuroblastoma cells are induced to differentiate by removal of serum, steps 1 and 2 take 24-48 hr, steps 3 and 4 take place 24 hr later, and step 5 continues for several days. Some Implications of the Movement and Aggregation of Initiation Sites The studies of neuroblastoma differentiation demonstrate that microtubule initiation sites can change their position within the cell. Such rearrangements appear to be closely related to neurite
Cell 262
INITIATION SITE AGGREGATION
UNDIFFERENTIATED
interest to know whether the different classes of initiation sites observed in epithelial and fibroblast cells (see Spiegelman et al., 1979) represent different aggregates of the same material or are chemically distinct structures. Knowledge of the nature of the initiation sites would facilitate our understanding of the regulation of microtubule assembly and the function of microtubules within cells.
1
m MICROTUBULE ORIENTATION
a
5
NEURITE EXTENSION
DIFFERENTIATED Figure
8. Model
C
for Neurite
Outgrowth
in Neuroblastoma
Cells
formation in neuroblastoma cells, and it is probable that they are important for changes in morphology in many other types of cell. Alterations in the position of initiation sites may also be important in other functional aspects of microtubules. For example, microtubules appear to be involved in regulating changes in the direction of movement of fibroblast cells (Gail and Boone, 1970; Gail and Boone, 1971) and are also required for the directed chemotactic movements of leukocytes (Caner, 1965; Malech, Root and Gallin, 1977). It seems probable that the ability of these cells to alter their direction of movement requires the rearrangement of microtubules. The observed mobility of microtubule initiation sites in neuroblastoma cells suggests that the movements of these sites could facilitate the rearrangements of microtubule networks in motile cells, enabling them to move in a directed manner. The ability of microtubule initiation sites to move and aggregate may also have implications for the assembly of the mitotic spindle. The spindle consists of many microtubules oriented around two poles, each of which contains a centriole. The theory that the centriole itself nucleates the assembly of spindle microtubules has recently been questioned (Pickett-Heaps, 1969, 1971; Berns and Richardson, 1977). It now seems probable that many microtubules insert into an electron-dense cloud of material surrounding the centriole. The movement and aggregation of microtubule initiation sites in neuroblastoma cells suggest that the electrondense cloud surrounding the centriole may actually represent an aggregate of some or all of the interphase initiation sites. Many questions regarding the microtubule initiation sites remain to be answered. It will be important to obtain information about the structure and composition of these sites. It would be of particular
Experimental
Procedures
N115 mouse neuroblastoma cells were a gift from M. Nirenberg (NIH, Bethesda, Maryland). Cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum in a 10% CO, atmosphere at 37°C. Cells were harvested by trypsinization, and 5 x IO5 ceils were allowed to attach onto 22 mm* coverslips in a 100 mm petri dish for at least 24 hr before each experiment. lmmunofluorescence with antibody to tubulin was performed by minor modifications of the cytoskeletal procedure of Osborn and Weber (1977) as described by Spiegelman et al. (1979). All other materials and methods are as described in the accompanying paper (Spiegelman et al., 1979). Acknowledgments We thank Marshall Nirenberg for providing the neuroblastoma cell line. We thank V. Kalnins and J. Connolly at the University of Toronto for providing the anti-tubulin serum. This work was supported by grants from the American Cancer Society and the National institute for General Medical Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received
August
18, 1978;
revised
October
5, 1978
References Augusti-Tocco, G. and Sato, G. (1969). Establishment tional clonal lines of neurons from mouse neuroblastoma. Nat. Acad. Sci. USA64, 311-315. Berns, M. W. and Richardson, S. M. (1977). mitosis after selective laser microbeam destruction lar region. J. Cell Biol. 75, 977-982.
of funcProc.
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Blume, A., Gilbert, F., Wilson, S., Farber, J.. Rosenberg, Nirenberg, M. (1970). Regulation of acetylcholinesterase roblastoma cells. Proc. Nat. Acad. Sci. USA67, 786-792.
R. and in neu-
Brinkley, B. R., Fuller, G. M. and Highfield, D. P. (1976). Tubulin antibodies as probes for microtubules in dividing and nondividing mammalian cells. In Cell Motility, R. Goldman, T. Pollard and J. Rosenbaum, eds. (New York: Cold Spring Harbor Laboratory), pp. 435-445. Caner, J. E. 2. (1965). tis Rheum. 8, 757-763.
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Daniels, M. (1975). The role of microtubules in the growth stabilization of nerve fibers. Ann. NY Acad Sci. 253, 535-544. Frankel, F. R. (1976). Organization of microtubules. Proc. Nat. Acad.
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Gail, M. H. and Boone, C. W. (1970). The locomotion fibroblasts in culture. Biophys. J. 70, 980-993. Gail, M. fibroblast Granholm, bules and 563-584.
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Isenberg, G., Rieske, E. and Kreutzberg, G. W. (1977). Distribution of actin and tubulin in neuroblastoma cells. Cytobios 75, 382-389. Malech, H. L., Root, R. V. and Gallin, J. I. (1977). Structural analysis of human neutrophil migration: centriole, microtubule and microfilament orientation and function during chemotaxis. J. Cell Biol. 75, 866-693. Nelson, P., Ruffner, W. and Nirenberg, cells with excitable membranes grown Sci. USA64, 1004-1010.
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Osborn, M. and Weber, K. (1976). Cytoplasmic microtubules in tissue culture cells appear to grow from an organizing structure toward the plasma membrane. Proc. Nat. Acad. Sci. USA 73, 867871. Osborn, M. and Weber, K. (1977). The display transformed cells. Cell 72, 561-571. Perry, M. M. and Waddington. blastoporal cells in the newt. 330.
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February
1979,
Copyright
0 1979 by MIT
Aggregation of Microtubule Initiation Sites Preceding Neurite Outgrowth in Mouse Neuroblaktoma Cells Bruce M. Spiegelman,* Margaret A. Lopatat Marc W. KirschnertS Department of Biochemical Sciences Princeton University Princeton, New Jersey 08540
and
Summary By examining microtubule regrowth using immunofluorescence with antibody to tubulin, we have studied the structure and intracellular localization of microtubule initiation sites in undifferentiated and differentiated mouse neuroblastoma cells. The undifferentiated cells are round and lack cell processes. They contain an average of 12 initiation sites per cell. Each of these sites, which are located near the cell nucleus, initiates the growth of several microtubules in a radial formation. In contrast to the undifferentiated cells, neuroblastoma cells stimulated to differentiate by serum deprivation are asymmetrical, containing one or two very long neurites. These cells have a single, large microtubule initiation center which can be visualized not only by immunofluorescence but by phase-contrast and differential interference microscopy as well. The initiation site measures 3-4 p in diameter and is located in the cell body along a line defined by the neurite. During cell differentiation, the large initiation center seems to be formed by the aggregation of many smaller sites. This process precedes neurite extension by about 24 hr. The growth of microtubules from this center appears to be highly oriented, since most microtubules initially grow into the neurite processes rather than into the cell interior. Thus major changes in the structure and location of microtubule initiation sites occur during the differentiation of neuroblastoma cells. Similar changes are likely to be involved in alterations in the morphology of other cell types. Introduction Changes in cell shape are important in embryonic development and cell differentiation. The arrangement of structural proteins, such as actin, microtubules and intermediate filaments, strongly influences if not determines cell morphology. We may therefore expect that changes in the intracellular distribution of these structural elements are a sigl Present address: Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139. t Present address: Department of Biochemistry and Biophysics, University of California School of Medicine, San Francisco, California 94143. $ To whom correspondence should be addressed.
nificant part of cell differentiation. For example, Perry and Waddington (1966) showed that microtubules are prominent components of the necks of the highly asymmetric bottle cells present at the blastopore in amphibians. They are also present in elongating cells in the primitive streak in birds (Granholm and Baker, 1970). Microtubules are particularly prominent in neural development. The highly asymmetric nerve processes are packed with dense parallel arrays of microtubules. Treatment of immature nerve cells with antimicrotubule drugs such as colchicine prevents neurite extension and causes withdrawal of previously extended neurites (Yamada, Spooner and Wessels, 1970; Seeds et al., 1970; Daniels, 1975), indicating that microtubules are required for both extension and maintenance of these cell processes. The mechanism by which the cell changes its microtubule distribution is unknown. However, since microtubules grow from distinct initiation centers (Frankel, 1976; Osborn and Weber, 1976; Brinkley, Fuller and Highfield, 1976; Spiegelman, Lopata and Kirschner, 1979), the precise location and structure of these centers may determine the microtubule distribution and hence the cell morphology. In the accompanying paper (Spiegelman et al., 1979), we have shown that mammalian fibroblasts and epithelial cells contain several initiation sites for microtubule growth. Three classes of sites could be distinguished and their arrangement and nature were found to differ in epithelial and fibroblast cells. In this study, we have sought to determine whether the position of these sites is closely related to cell morphology and whether changes in initiation sites accompany cellular differentiation. We have compared the structure and distribution of these sites in neuroblastoma cells before, during and after stimulation of neurite outgrowth. The results show that initiation sites alter their position and coalesce before the process of neurite outgrowth, suggesting that changes in their arrangement may control neurite formation. Results lmmunofluorescence of Neuroblastoma Cells with Antibody to Tubulin When grown in culture medium containing 10% serum, mouse neuroblastoma cells (line N11.5) are small rounded cells, 30-50 p in diameter, having few extensions or processes (Figure IA). Removal of serum causes cell differentiation (Seeds et al., 1970). Figure 1C shows the effect of 5 days of incubation in culture medium lacking serum. Approximately 80-90% of the cells have extended neurites which may be as long as 500 p. Most of
Cell 254
the cells contain a single neurite, but we occasionally observed cells with two neurites, such as one of those shown in Figure 1C. Accompanying this morphological change induced by serum deprivation are a number of enzymatic and electrical changes (Augusti-Tocco and Sato, 1969; Nelson, Ruffner and Nierenberg, 1969; Blume et al., 1970), which suggest that this system may be a model for nerve cell differentiation. To visualize microtubule networks in the neuroblastoma cells, we have stained cells with antibody to tubulin with the same cytoskeleton preparation technique used in our previous study of fibroblast and epithelial cells (Spiegelman et al., 1979, adapted from Osborn and Weber, 1977). lmmunofluorescence staining of undifferentiated cells and of cells induced to differentiate by serum deprivation is shown in Figures 1 B and lD, respectively. In both the undifferentiated and differentiated cells, intense staining is observed throughout the cells. It appears that the interphase microtubule network is so thick that it is difficult to resolve individual microtubules. Only in exceptionally flat cells can distinct microtubules be seen at the cell periphery (see also Osborn and Weber, 1977; Isenberg, Rieske and Kreutzberg, 1977). Microtubule Regrowth in Undifferentiated Neuroblastoma Cells As we observed in fibroblast and epithelial cells (Spiegelman et al., 1979), microtubule initiation sites in neuroblastoma cells are readily visualized during the process of microtubule regrowth after exposure to depolymerizing agents. If cells are exposed to colcemid for 90 min at 0.4 pg/ml, no microtubule structures are visible. When cells are exposed to colcemid for 35 min and then allowed to recover for 25 min in fresh medium, several bright points of fluorescence are visible in the interior of the cell, as shown in Figure 2A. Figure 2B shows the phase-contrast image of the same cell. We could not identify any distinct structures in phase-contrast which correspond to the bright fluorescent points. After IO min of further incubation in the absence of the drug, microtubules grow extensively from several sites. In Figure 2C, nine sites are visible surrounding the cell nucleus. Figure 3 is a higher magnification photograph of another undifferentiated cell, showing regrowth from 14 distinct sites in the perinuclear region. The microtubules appear to grow out in all directions from the individual initiation points. After 50 min of recovery, microtubules have grown throughout the cell, although the initiation sites are still discernible, as shown in Figure 2D. After this time, individual microtubules are difficult to resolve, presumably because there is a superposition of dense
arrays of microtubules. We find that undifferentiated neuroblastoma cells contain an average of 12.3 i- 4.5 initiation sites per cell, with a range of 6-27. in 43 cells. Microtubule Regrowth in Differentiated Neuroblastoma Cells There are special problems associated with observing microtubule regrowth in differentiated neuroblastoma cells. As Seeds et al. (1970) showed, cellular processes are extremely sensitive to antimicrotubule drugs. We have found, for example, that exposure of cells to colcemid at 0.4 pglml for 90 min is sufficient to cause complete absorption of neurites into the cell body and eliminates immunofluorescent staining of any discrete microtubule structures. Very short exposures, however, will achieve nearly complete microtubule depolymerization without neurite retraction. Since we have found that microtubule depolymerization continues for about 25 min after the removal of colcemid, we have minimized the treatment times by exposing cells for a period short enough so that depolymerization approaches completion only during the recovery period. Thus the length of time the cell is devoid of its microtubule network is kept to a minimum. When differentiated neuroblastoma cells are exposed to 0.4 pg/ml of colcemid for 35 min, followed by 25 min of recovery in fresh medium, the neurites are retained, but the bright fluorescence disappears from the neurite and most of the cell body. A single fluorescent center, however, is visible in these cells (Figure 4A). In virtually all cases, this center is found in the cell body along a line defined by the neurite extension. It occurs most commonly between the neurite and the cell nucleus but occasionally can be found directly on top of the cell nucleus. Although it is difficult to see in a photograph taken at a single focal plane, this structure has an irregular scalloped appearance and seems to be an aggregate of a number of fluorescent points. After 10 min of further incubation in colcemid-free medium, short microtubules can be seen emanating in many directions in a roughly radial fashion from the large fluorescent center (Figure 4C). Unlike fibroblasts, neuroblastomas are thick cells and the microtubules do not all lie in the same plane of focus, making them more difficult to photograph. Figure 4D shows that after 50 min in colcemid-free medium, the microtubules have elongated into the neurite. At this stage of regrowth, the microtubules seem oriented, since less growth extends into the cell body than into the neurite. After longer periods of recovery, the intensity of fluorescence increases throughout the cell and the neurite, and the detailed microtu-
Aggregation 255
Figure
of Microtubule
1. lmmunofluorescence
Initiation
Sites
of Mouse
Neuroblastoma
Cells with Antibody
to Tubulin
Cells were grown on glass coverslips and induced to differentiate by 5 days of serum immunofluorescence by the cytoskeletal procedure of Osborn and Weber (1977) as described (A) (275X) phase-contrast image of undifferentiated cells: (B) (770X) immunofluorescence contrast image of differentiated cells; (D) (770X) immunofluorescence of a differentiated cell.
starvation. They were then processed for by Spiegelman et al. (1979). Bar = IO p. of undifferentiated cells: (C) (275X) phase-
Cell 256
Figure
2. Regrowth
of Microtubules
in Undifferentiated
Neuroblastoma
Cells
after Colcemid
Treatment
Cells grown on coverslips were treated with 0.4 pg/ml colcemid for 35 min, rinsed 3 times with immunofluorescence at the indicated times after the removal of colcemid. Bar = IO CL. (A) (770X) 25 min; (6) (770X) phase-contrast of same cell as (A); (C) (770X) 35 min; (D) (770X) 50 min.
fresh
medium
and
processed
for
Aggregation 257
Figure
of Microtubule
3. Higher
Ceils were
treated
Magnification as described
Initiation
Sites
Photograph in the legend
of Microtubule to Figure
Regrowth
2. Recovery
bule array is obscured, as in Figure 1D. In over 1000 cells examined, those containing neurite processes always contained asingle large initiation center. An image corresponding to the large microtubule initiation center can be observed with phase-contrast and differential interference microscopy in cytoskeletal preparations of differentiated neuroblastoma cells. An initiation structure is shown in Figure 4A with immunofluorescence. The corresponding structure is shown in the phase-contrast photograph of the same cell in Figure 4B, where it appears as a single, irregularly shaped dark object 3-4 p in diameter. In Figure 5B, a differentiated neuroblastoma cell is shown by differential interference microscopy. The initiation center appears to be a roughly circular structure 3-4 p in diameter, adjacent to the cell nucleus, which corresponds in size and position to the initiation structure stained with the fluorescent antibody (Figure 5A). The differential interference image shows that the initiation center is nonuniform and appears to be composed of several discrete components. The size and shape of the initiation center seen by
in Undifferentiated after
the removal
Neuroblastoma of colcemid
Ceils
was for 35,min
(1760X).
Bar = IO p.
phase-contrast and differential interference microscopy remain unchanged throughout the initial stages of microtubule regrowth. About 10% of the differentiated neuroblastoma cells contain two neurite processes. These cells also contain only a single large microtubule initiation center. Figure 6 shows a cell with two neurites which was allowed to recover for 50 min after treatment with colcemid. The large fluorescent center is located above the nucleus. Microtubule regrowth is bidirectional in this case, with most of the microtubules growing into the two neurites. Very few microtubules grow perpendicular to a line connecting the two neurites and the initiation structure. We observed similar patterns of regrowth to those observed with colcemid after treatment with vinblastine in both differentiated and undifferentiated cells. Our attempts to examine microtubule regrowth in neuroblastoma cells after griseofulvin treatment have been unsuccessful. In differentiated cells, we could not achieve sufficient depolymerization without the neurites regressing into the cell body. In undifferentiated cells, the background
Cell 258
Figure 4. Regrowth of Microtubules Differentiated Neuroblastoma Cells
after
Colcemid
Cells were grown on coverslips in serum-free Theywere then treated with 0.4 kg/ml colcemid 3 times with fresh medium and processed cence at the indicated times after the removal IO/L. (A) (578X) 25 min; (B) (578X) phase-contrast (C) (578X) 35 min; (D) (578X) 50 min.
Treatment
in
medium for 3 days. for35 min, rinsed for immunofluoresof colcemid. Bar = of same
cell as (A);
staining after griseofulvin treatment was too tense to allow clear examination of regrowth.
in-
Time Dependence of Initiation Site Aggregation and Neurite Outgrowth It is not clear from these immunofluorescence experiments whether the formation of the single large initiation center in the differentiated neuroblastoma cells precedes or follows the extension of
Figure 5. Differential Interference rescence of an Initiation Center Cells
Microscopy in Differentiated
and ImmunofluoNeuroblastoma
Cells were treated as described in the legend to Figure 4 and photographed 35 min after the removal of colcemid. Bar = 10 CL. (A) (1160X) immunofluorescence; (6) (1160X) differential interference image of the same cell as (A).
neurites. To answer this question, we have followed the time course of neurite outgrowth and the formation of single large sites in the same cultures. Undifferentiated neuroblastoma cells were initially
Aggregation
of Microtubule
Initiation
Sites
259
Table 1. Time Course of Appearance Aggregation of Initiation Sites
of Neurites
and
(A)
(‘3
CC)
P)
W
Days after Removal of Serum
Cells with Neurites
Dispersed Sites
Aggregated Sites
Single Large Center
w
w
(“W
(“4
0
2
90
0.5
9.5
1
7
05
6
9.0
2
8
35
33
3
58
18
10
72
4
76
12
16
72
5
81
4
6
90
32
Cells on coverslips were grown in serum-free medium for the indicated length of time and examined for the presence of neurites of any length by phase-contrast microscopy. The same coverslips were then processed for immunofluorescence by the cytoskeletal procedure after treatment of cells with 0.4 wg/ml colcemid and 35 min of recovery incubation in fresh medium at 37°C. Cells which had a single large microtubule initiation structure were scored. Cells which contained aggregates of three or more distinct initiation sites, but which had not formed a single large initiation structure, were scored as “aggregated.” For each time point, 250-300 cells were examined.
Figure 6. Regrowth Two Neurites
of Microtubules
Cells were treated as described processed for immunofluorescence colcemid (770X). Bar = 10 /I.
in a Neuroblastoma
Cell with
in the legend to Figure 4 and 50 min after the removal of
grown on coverslips in medium containing 10% fetal calf serum, placed in serum-free medium and then examined at various times for neurite formation. The cells showing neurites of any size were scored. These cells were then treated with colcemid for 35 min, allowed to recover for 35 min and processed for immunofluorescence to visualize the initiation centers. We noted the number of cells with single initiation centers and examined whether these cells had neurites. For each time point, 250-300 cells were examined. As shown in Table 1, only 1.8% of the neuroblastoma cells grown in medium containing serum had neurites, while 9.5% had single initiation centers. At all time points, every cell with neurites had a single initiation center. After 1 day without serum, the number of cells with neurites grew to 7%, while the number of cells with single initiation centers remained about the same at 9%. At 2 days, there was a greater than 3 fold increase in the proportion
of cells with single initiation centers to 32%, while the percentage of cells with neurites increased only slightly to 8%. At 3 days, there was a large proliferation of cells with neurites, increasing 7 fold to 58%, and an increase in the number of cells with single centers to 72%. Thereafter the percentage of cells with neurites and cells with single centers increased more slowly. In all cases, the number of cells with neurites was a subset of the cells with single initiation centers. Although one must be cautious in interpreting kinetic data, the analysis of initiation sites and neurites is clearly inconsistent with the notion that neurite outgrowth precedes the formation of a single large center. At 2 days, 4 times as many cells had single large centers as had neurites and with time this difference narrowed. There were no clear examples of cells with neurites that did not have a single initiation center. A replotting of columns B and E of Table 1 (not shown) shows that the time course of appearance of neurites is displaced about 24 hr after the appearance of single initiation sites. Intermediates in Initiation Site Aggregation The kinetic data suggest that the appearance of single large initiation centers precedes neurite formation. They do not, however, suggest how this occurs. We have surveyed the neuroblastoma cells during serum deprivation and have noted that in
Cell 260
reaching 90% at 5 days. This behavior suggests that small aggregates of initiation sites are intermediates in the formation of single, large complex centers.
cultures deprived of serum for 2 days, many cells contain multiple initiation sites which appear to be in the process of aggregating. Three such cells, which lack neurites but show initiation in various stages of aggregation, are shown in Figure 7. The appearance of the initiation centers in these cells should be contrasted with the typical dispersed appearance of the initiation sites in undifferentiated cells in Figures 2 and 3. In Table 1, we have distinguished among cells containing dispersed sites (column C), cells containing aggregates of at least three initiation sites (column D) and cells containing a single large initiation center (column E). At time zero, 90% of the cells had dispersed sites; a very small percentage (0.5%) had aggregated sites, while 9.5% had single, large centers. By the second day of serum deprivation, about one third of the cells had dispersed sites, one third had aggregated sites and one third had single initiation centers. Thereafter the number of cells containing aggregated sites dropped to 6% at day 5, while the number of cells with single centers increased to 90%. Thus small aggregates of initiation sites occur in a large portion of cells at 2 days, but are less evident at 0 and 5 days. At the same time, the number of cells with single centers increases markedly at 2 and 3 days,
Figure
7. Intermediates
Cells grown min recovery
on coverslips incubation
in the Aggregation were incubated in fresh medium,
of Microtubule
Initiation
Discussion In the accompanying paper (Spiegelman et al., 1979) we showed that mammalian epithelial and fibroblast cells have several initiation sites for microtubule assembly within each cell. This paper examines the number and nature of these sites during a developmental process thought to involve microtubule assembly. Although many cellular differentiation events have been shown to involve microtubules, the study of neurite outgrowth in neuroblastoma cells has particular advantages. Neurite outgrowth depends completely upon the assembly of microtubules. In addition, neuroblastoma differentiation is readily initiated in culture, takes place in single cells rather than in whole tissues and seems to be related to the normal process of nerve growth (Seeds et al., 1970). Changes in Initiation Sites during Differentiation Undifferentiated neuroblastoma cells have several (12 +- 4) sites for microtubule initiation in each cell.
Sites
in serum-free medium for 2 days. After treatment with 0.4 pg/ml colcemid they were processed for immunofluorescence (770X). Bar = 10 CL.
for 35 min and a 35
Aggregation 261
of Microtubule
Initiation
Sites
These multiple sites are observed when visualized after depolymerization by colcemid or vinblastine. They are rather uniformly arranged around the cell nucleus and there is no apparent clustering of sites. Similarly, we observed no clustering of initiation sites in fibroblast or epithelial cells (Spiegelman et al., 1979). None of these cell types is highly polarized or asymmetric and all contain uniformly arranged initiation sites, suggesting that the arrangement of these sites may be related to the overall cell morphology. In contrast to the undifferentiated cells, the differentiated neuroblastoma cells are highly asymmetric, possessing one or sometimes two long neurites. Differentiated cells have only one initiation center per cell, which is located near the cell nucleus, directly on a line from the neurite process. We observed a single initiation center in all cells which had neurite processes. However, not all cells which had single initiation centers had neurites. As shown in Table 1, 2% of the undifferentiated cells had neurites but 7% had single initiation centers. The disparity is even more striking 2 days after the removal of serum, when 8% of the cells had neurites and 32% had single initiation centers. Thus it seems very improbable that single large initiation centers could be caused by neurite formation, and more plausible that neurite production was a consequence of the formation of single initiation sites. This problem was studied more extensively by examining the time course of neurite outgrowth and the aggregation of initiation sites. The time course experiments in Table 1 demonstrate that the formation of a single large center precedes neurite formation and suggests that the formation of this large center occurs by aggregation of the dispersed sites. The fraction of cells with neurites and the fraction of cells with single centers both increase monotonically with time, while the fraction of cells with dispersed sites decreases monotonically. A plot of the data indicates that neurite extension lags about 24 hr behind the formation of single initiation centers. The fraction of cells with aggregated initiation sites shows the behavior of a kinetic intermediate, increasing from 0.8 to 33% from 0 to 2 days, and thereafter decreasing to 6% 5 days after the removal of serum. The peak in the fraction of cells with aggregated sites occurs before the rapid increase in cells with neurites. It was possible to see some structure in the large initiation center using immunofluorescence, although the structure was difficult to illustrate by photographs taken at a single plane of focus. The initiation center appears to be an irregular mass which might represent an aggregate of individual sites. Phase-contrast microscopy of cytoskeletal
preparations shows a dense body having the same position, size and shape as that seen by immunofluorescence (see Figures 4A and 4B). The differential interference image of this center in Figure 58 also gives the impression of a compound structure about 3 p in diameter. There is probably little more that can be learned about the structure of the large initiation center by light microscopy: further information will certainly require electron microscopic investigations. There are several reasons to suspect that neurite extension requires one or more steps in addition to the aggregation of microtubule initiation sites. First, there is a significant time lag of about 24 hr between the two processes, which allows the observation of many cells lacking neurites but containing aggregated initiation sites. Such cells would be expected to be rare if the aggregation of initiation sites was the last step or the rate-limiting step in neurite formation. Second, the first microtubules to regrow from the initiation center in differentiated cells seem to be directed into the neurite. This result is in contrast to the undirected growth from the dispersed sites in undifferentiated cells (Figures 4 and 6), and suggests that there must be a mechanism for orienting microtubules as well as a mechanism for localizing their points of initiation. This could be accomplished in several ways, such as assembly of the initiation center in a manner which allows microtubule growth in only one direction. Alternatively, there may be localized structures on the cell membrane which would stabilize certain microtubules or promote their assembly. A model for neurite outgrowth in neuroblastoma cells summarizing these observations is shown in Figure 8. The undifferentiated cell contains several initiation sites dispersed around the cell nucleus. Steps 1 and 2 illustrate the migration of initiation sites into one part of the cell and their aggregation into a single complex center. In step 3, microtubules are oriented, through some unknown mechanism, so that they grow primarily in a single direction toward the cell membrane. In steps 4 and 5, neurites grow out from the cell body and elongate. This process depends upon the polymerization of microtubules. If neuroblastoma cells are induced to differentiate by removal of serum, steps 1 and 2 take 24-48 hr, steps 3 and 4 take place 24 hr later, and step 5 continues for several days. Some Implications of the Movement and Aggregation of Initiation Sites The studies of neuroblastoma differentiation demonstrate that microtubule initiation sites can change their position within the cell. Such rearrangements appear to be closely related to neurite
Cell 262
INITIATION SITE AGGREGATION
UNDIFFERENTIATED
interest to know whether the different classes of initiation sites observed in epithelial and fibroblast cells (see Spiegelman et al., 1979) represent different aggregates of the same material or are chemically distinct structures. Knowledge of the nature of the initiation sites would facilitate our understanding of the regulation of microtubule assembly and the function of microtubules within cells.
1
m MICROTUBULE ORIENTATION
a
5
NEURITE EXTENSION
DIFFERENTIATED Figure
8. Model
C
for Neurite
Outgrowth
in Neuroblastoma
Cells
formation in neuroblastoma cells, and it is probable that they are important for changes in morphology in many other types of cell. Alterations in the position of initiation sites may also be important in other functional aspects of microtubules. For example, microtubules appear to be involved in regulating changes in the direction of movement of fibroblast cells (Gail and Boone, 1970; Gail and Boone, 1971) and are also required for the directed chemotactic movements of leukocytes (Caner, 1965; Malech, Root and Gallin, 1977). It seems probable that the ability of these cells to alter their direction of movement requires the rearrangement of microtubules. The observed mobility of microtubule initiation sites in neuroblastoma cells suggests that the movements of these sites could facilitate the rearrangements of microtubule networks in motile cells, enabling them to move in a directed manner. The ability of microtubule initiation sites to move and aggregate may also have implications for the assembly of the mitotic spindle. The spindle consists of many microtubules oriented around two poles, each of which contains a centriole. The theory that the centriole itself nucleates the assembly of spindle microtubules has recently been questioned (Pickett-Heaps, 1969, 1971; Berns and Richardson, 1977). It now seems probable that many microtubules insert into an electron-dense cloud of material surrounding the centriole. The movement and aggregation of microtubule initiation sites in neuroblastoma cells suggest that the electrondense cloud surrounding the centriole may actually represent an aggregate of some or all of the interphase initiation sites. Many questions regarding the microtubule initiation sites remain to be answered. It will be important to obtain information about the structure and composition of these sites. It would be of particular
Experimental
Procedures
N115 mouse neuroblastoma cells were a gift from M. Nirenberg (NIH, Bethesda, Maryland). Cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum in a 10% CO, atmosphere at 37°C. Cells were harvested by trypsinization, and 5 x IO5 ceils were allowed to attach onto 22 mm* coverslips in a 100 mm petri dish for at least 24 hr before each experiment. lmmunofluorescence with antibody to tubulin was performed by minor modifications of the cytoskeletal procedure of Osborn and Weber (1977) as described by Spiegelman et al. (1979). All other materials and methods are as described in the accompanying paper (Spiegelman et al., 1979). Acknowledgments We thank Marshall Nirenberg for providing the neuroblastoma cell line. We thank V. Kalnins and J. Connolly at the University of Toronto for providing the anti-tubulin serum. This work was supported by grants from the American Cancer Society and the National institute for General Medical Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received
August
18, 1978;
revised
October
5, 1978
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